| Objective:The agrC specific binding polypeptide sequence was obtained by gene recombination and phage peptide library method in the previous study and then we will study the mechanism of action of Staphylococcus epidermidis agrC specific binding polypeptide on the biofilm formation of Staphylococcus epidermidis on the surface of biomaterials.It lays the foundation for the specific polypeptide drugs for the treatment of Staphylococcus epidermidis biofilm formation related infections.Methods:1.According to different final concentration of agrC specific binding polypeptide(N1)and irrelevant peptide(NO)(100?g/ml,200?g/ml,400?g/ml,800?g/ml,1600?g/ml),it was divided into experimental group and control group.The experimental group N1 polypeptide was set up 6 concentration gradient group,the control group NO polypeptide was set up 6 concentration gradient group.The 96-well plate of PVC material was used as the bacterial biofilm attachment carrier,and the standard strain of Staphylococcus epidermidis(ATCC35984 biofilm phenotype positive strain,ATCC12228 biofilm phenotype negative strain)was used as the experimental bacteria for cultivating 24 hours.The semi-quantitative detection method for crystal violet was used to evaluate the ability to form bacterial biofilm,so that determine the optimal inhibitory concentration of N1 polypeptide.2.According to the addition of the polypeptide,the agrC specific binding polypeptide(Nl)was used as the experimental group and the irrelevant peptide(NO)was used as the control group,and the sterilized water was used as the blank control group(The final concentration of peptides in Nl group and NO group was the best inhibitory concentration determined in the previous period,and the blank group was added with the same amount of sterilized water).The standard strain of Staphylococcus epidermidis(ATCC 35984 biofilm phenotype positive strain)was selected as the experimental bacteria.After each group correspondingly added in N1 polypeptide,NO polypeptide and sterilized water and then cultivated for 6h,12h,18h,24h,30h,?XTT colorimetry was used to detect the growth kinetics of Staphylococcus epidermidis;?Quantitative Real time PCR was used to detect the the change of expression of Staphylococcus epidermidis biofilm-related genes(AtlE,icaA,fbe,icaR):?the method of phenol-sulfate was used the analytical pure glucose standard to make a standard curve,and then determining the optical density(OD)value of Staphylococcus epidermidis at 490 nm to calculate the PIA content closely related to the biofilm formation of Staphylococcus epidermidis.;? PRM relative quantitative proteomics was used to determine the content of short peptide PSMs associated with Staphylococcus epidermidis virulence factors and biofilm dissemination.Results:1.With the final concentration of agrC specific binding polypeptide increased,the inhibitory effect on biofilm formation ability was enhanced.When the final concentration of agrC specific binding polypeptide was 800?g/ml,the biofilm formation ability was the weakest(OD mean 0.901),the difference was statistically significant(P<0.05);When the final concentration of agrC specific binding polypeptide continues to increase to 1600 ?g/ml,the ability to form bacterial biofilm was no longer weakened.(OD mean is 1.127).It indicated that the N1 polypeptide with 800?g/ml was the best effective inhibitory concentration,and the antibacterial effect was no longer strengthened with the concentration continued to increase.2.XTT colorimetry detection showed that in the 6h,the growth kinetics of bacterial biofilm in N1 group(OD mean 0.317)was significantly higher than that in NO group(OD mean value 0.167)and sterilized water group(OD mean value 0.204),the difference was statistically significant(P<0.05);In the 12h,the growth kinetics of bacterial biofilm in N1 group(OD mean value 0.124)was significantly lower than that in NO group(OD mean value 0.288)and sterilized water group(OD mean value 0.264),the difference was statistically significant(P<0.05).There was no significant difference in biofilm growth kinetics between N1 group,NO group and sterilized water group at 18h,24h and 30h(P>0.05).It indicated that the inhibition of biofilm formation by N1 polypeptide was mainly in the stage of biofilm formation accumulation stage.3.Quantitative Real-time PCR showed that in the 12h the expression level of atlE,icaA,fbe and icaR in N1 group were down-regulated compared with NO group and sterilized water group,the difference was statistically significant(P<0.05).At 6h,18h,24h and 30h,the expression level of atlE,icaA,fbe and icaR in N1 group were up-regulated compared with NO group and sterilized water group,the difference was statistically significant(P<0.05).It indicated that the N1 polypeptide can down-regulate the biofilm forming genes atlE,icaA and fbe during the biofilm formation accumulation stage,while the biofilm inhibitory gene icaR can be up-regulated in other stages.4.The content of PIA in bacterial biofilm increased with time by phenol-sulfate method.The content of PIA in N1 group,NO group and sterilized water group was the same in the 6h,there was no statistical difference(P>0.05).in the 12h,the PIA content in N1 group(average PIA content of 0.00190mg)was lower than that in NO group(PIA content average 0.00543mg)and sterilized water group(PIA content average 0.00563mg),the difference was statistically significant(P<0.05);The content of PIA in N1 group,NO group and sterilized water group was the same at 18h,24h and 30h,there was no statistical difference(P>0.05).It indicated that the N1 polypeptide can inhibit PIA synthesis during the accumulation stage of biofilm formation.5.The PSMs content of PRM relative quantitative proteomics was compared in different groups in different time:in the 6h,the N1 group was higher than the NO group and the sterilized water group;in the 12h,the N1 group was higher than the NO group and the sterilized water group;in the 18h,the N1 group was higher than the NO group and sterilized water group;in the 24h,the N1 group was higher than the NO group,which was lower than the sterilized water group;in the 30h,N1 group was lower than the NO group and the sterilized water group.It indicated that N1 polypeptide can inhibit the synthesis of PSMs during the maturation stage and dissemination stage of biofilm formationConclusions:1.Staphylococcus epidermidis agrC specific binding polypeptide inhibits Staphylococcus epidermidis biofilm formation with the concentration-effect relationship.When a certain concentration(800 p.g/ml)was reached,the inhibition of Staphylococcus epidermidis biofilm formation was the most significant.2.In the accumulation stage of Staphylococcus epidermidis biofilm,'formation,Staphylococcus epidermidis agrC specific binding polypeptide down-regulates the expression of membrane forming genes atlE,icaA and fbe by inhibiting the activation of staphylococcus epidermidis agr quorum sensing system;then in the maturation stage and dissemination stage of biofilm formation it may inhibit the synthesis of PSMs.This may be the main mechanism of agrC specific binding polypeptides that it inhibit Staphylococcus epidermidis biofilm formation on the surface of biomaterials. |
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